摘要
目的 预测宫颈癌细胞中人类与鼠同源乳腺转化基因(HMATI)的功能及筛选出能有效沉默该基因的干扰小RNA。方法 采用细胞原癌基因、抑癌基因分类芯片对原发性宫颈癌标本进行筛选,通过生物信息学分析HMAT1 基因,针对HMAT1基因设计了三条特异性干扰小RNA,分别与pRNAU6.1 Hygro重组后,与含有该基因的重组体瞬时共转染B16F0细胞,用逆转录聚合酶链反应(RT PCR)技术检测对HMAT1基因的干扰效果。结果 细胞原癌基因、抑癌基因分类芯片筛选出表达量明显上调的基因共四条。对其中上调明显的HMAT1基因,通过生物信息学预测其为一个调节细胞生长的看家基因,其编码的蛋白质为一个Ⅰ型跨膜蛋白,且其酪氨酸蛋白激酶Ⅱ磷酸化位点可能与HMAT1 蛋白功能活化有关。半定量检测结果表明其中第一条特异性干扰小RNA有显著性抑制效果(P<0.05)。结论 初步预测HMAT1 基因很可能是与宫颈癌相关的癌基因,其编码的蛋白质可能参与细胞的信号转导,设计出针对该基因的干扰小RNA可以有效沉默HMAT1基因。
Objective To predict the function of human homolog of mouse MAT1 oncogene and screen out the small interfering RNA that can effect ively silence the HMAT1 gene.Methods After microarray analysis on the sample of cervical car cinoma, the up-regulated HMAT1 gene was analyzed by bioinformatical methods on both nucleic acid and amino levels. Then recombinant plasmids were constructed w hich can produce small interfering RNAs (siRNAs) to silence the HMAT1 gene and t he results were detected by semi- quantitative RT- PCR technique. Results There were four up-regulated genes found expressed by the study of gene chips. The biological information shows that the HMAT1 gene mi ght regulate the growth of cells. The protein encoded by HMAT1 gene might be a m ember of transmembrane protein Ⅰ. Two casein kinase Ⅱ phophorylation sites of HMAT1 gene might be related to the activation of HMAT1 protein. For the first si RNA, when the quatity of two recombined plasmids were well-proportioned, the ex perimental group was significantly different with the control group (P< 0.0 5), But the group of unspecific siRNA had no significantly difference with the control group (P> 0.05). Conclusion HMAT1 gene might be an oncogene related to cervical cancer and the protein encoded by it might be participate in the signal transduc tion.The certain siRNA can efficiently silence the HMAT1 gene.
出处
《肿瘤》
CAS
CSCD
北大核心
2005年第2期113-117,共5页
Tumor
基金
国家自然科学基金资助项目(编号:30171042
30250008)
